Cellular regulation of nitric oxide synthase expression and S-nitrosylation

一氧化氮（NO）是一种生物活性气体，控制着广泛的细胞生物过程。它主要通过引起靶蛋白s -亚硝基化来发挥其生物学作用，这是在靶半胱氨酸上共价添加亚硝基片段，是细胞中酶控制的过程。s -亚硝基化控制蛋白质功能，并被认为在细胞生物学中具有与蛋白质磷酸化相似的重要性。然而，对其调控和生物学效应的研究相对较少。由于NO的生物学重要性，我的研究计划的长期目标是了解这种气体的生物学效应和细胞调节。为了充分了解一氧化氮的生物学特性，研究其产生的调控机制是很重要的。一氧化氮由一氧化氮合酶（nos）合成，其中有三种亚型：神经元型（nNOS）、诱导型（iNOS）和内皮型（eNOS）。细胞因子可调节所有nos的表达，我们研究了细胞因子对iNOS和eNOS表达的调控。关于iNOS，我们已经证明NO启动一个正反馈回路，通过诱导Ras s -亚硝基化来放大iNOS的表达。我们还知道，no介导的iNOS表达扩增是通过增加mRNA翻译发生的。然而，我们不知道Ras s -亚硝基化是如何在细胞中被调节的，以及我们已经确定的no介导的反馈通路是如何控制iNOS mRNA翻译的。这些问题需要调查。除了iNOS，我们还确定细胞因子IL-17通过一种未知的翻译后机制上调eNOS的表达。基于我们的研究结果，本发现基金的短期目标是确定Ras s -亚硝基化的细胞调控以及iNOS和eNOS表达的转录后调控。这项工作将促进我们对NO的细胞生物学效应和NOS调控的理解。目的1：确定Ras s -亚硝基化的调控机制及其对Ras功能的影响。我们已经确定inos衍生的NO导致Ras s -亚硝基化，但不知道发生这种情况的机制。细胞中发生的大部分s -亚硝基化是由催化反应的亚硝基化酶和逆转反应的去亚硝基化酶进行酶调节的。我们将识别靶向Ras的亚硝基酶和去亚硝基酶。s -亚硝基化对Ras功能的影响也将通过检查这种蛋白质修饰对Ras与信号分子的关联和Ras定位的影响来确定。目的2：确定NO扩增iNOS mRNA翻译的机制。我们已经描述了一种新的no介导的信号通路，它可以放大iNOS mRNA的翻译，但不知道具体的翻译机制。我们将通过鉴定受NO影响的iNOS mRNA结合蛋白来确定这一点。候选蛋白的功能将通过用siRNA抑制其表达和改变其在iNOS mRNA序列中的RNA结合基序来确定。目的3：确定IL-17增加eNOS蛋白水平的翻译后机制。我们已经证明IL-17通过翻译后过程增加eNOS蛋白水平，但不知道其机制。IL-17对eNOS翻译后修饰的影响，已知会影响蛋白质稳定性，如磷酸化和泛素化，将通过蛋白质组学分析确定。候选蛋白修饰的作用将通过修饰位点的突变来确定。

Nitric oxide (NO) is a bioactive gas that controls a wide array of cell biological processes. It exerts its biological effects mainly by causing targeted protein S-nitrosylation, which is the covalent addition of a nitroso moiety onto target cysteines and is an enzymatically-controlled process in cells. S-nitrosylation controls protein function and has been proposed to be of similar importance to cell biology as protein phosphorylation. However, its regulation and biological effects are relatively understudied. Because of the biological importance of NO, the long-term goal of my research program is to understand the biological effects and cellular regulation of this gas. In order to fully understand the biology of NO, it is important to also study the mechanisms by which its production is regulated. NO is synthesized by enzymes called nitric oxide synthases (NOSs), of which there are three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Cytokines modulate the expression of all NOSs, and we have studied the cytokine regulation of iNOS and eNOS expression. With regard to iNOS, we have shown that NO initiates a positive feedback loop that amplifies iNOS expression by inducing Ras S-nitrosylation. We also know that NO-mediated amplification of iNOS expression occurs through increased mRNA translation. However, we do not know how Ras S-nitrosylation is regulated in cells and how iNOS mRNA translation is controlled by the NO-mediated feedback pathway that we have identified. These questions need to be investigated. In addition to iNOS, we have also determined that the cytokine IL-17 up-regulates eNOS expression through an unknown post-translational mechanism. Based on our findings, the short-term objectives of this Discovery Grant are to define the cellular regulation of Ras S-nitrosylation and post-transcriptional regulation of iNOS and eNOS expression. The work proposed will advance our understanding of the cell biological effects of NO and of NOS regulation. The three aims are: Aim 1: Determine the mechanism controlling Ras S-nitrosylation and its effect on Ras function. We have determined that iNOS-derived NO leads to Ras S-nitrosylation but do not know the mechanism by which this occurs. Much of the S-nitrosylation that occurs in cells is enzymatically-regulated by nitrosylases, which catalyze the reaction, and by de-nitrosylases, which reverse it. We will identify the nitrosylases and de-nitrosylases that target Ras. The effect of S-nitrosylation on Ras function will also be determined by examining the effect of this protein modification on the association of Ras with signaling molecules and on Ras localization. Aim 2: Determine the mechanism by which NO amplifies iNOS mRNA translation . We have characterized a new NO-mediated signaling pathway that amplifies iNOS mRNA translation but do not know the specific translational mechanisms involved. We will determine this by identifying iNOS mRNA binding proteins that are affected by NO. The function of candidate proteins will then be determined by inhibiting their expression with siRNA and by mutating their RNA binding motifs in iNOS mRNA sequences. Aim 3: Determine the post-translational mechanism by which IL-17 increases eNOS protein levels. We have shown that IL-17 increases eNOS protein levels through a post-translational process but do not know the mechanism. The effect of IL-17 on eNOS post-translational modifications that are known to affect protein stability, such as phosphorylation and ubiqutinylation, will be determined by proteomic analysis. The role of candidate protein modifications will then be determined by mutating the modification site(s).